Expression of SeMet labeled proteins: Difference between revisions

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1475 ml ddH<sub>2</sub>O
1475 ml ddH<sub>2</sub>O
400 ml M63 stock salts
400 ml M63 stock salts
20 ml of solution 1
 
20 ml of [http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php?title=Expression_of_SeMet_labeled_proteins&action=submit#solution_1: solution 1]
 
20 ml of solution 2
20 ml of solution 2
20 ml of solution 3
20 ml of solution 3
20 ml of solution 4  
20 ml of solution 4  
20 ml of solution 5
20 ml of solution 5
2 ml of 1M MgSO<sub>4</sub> stock
2 ml of 1M MgSO<sub>4</sub> stock
20 ml of 20% glucose stock
20 ml of 20% glucose stock
8 ml of ampicillin stock (100 mg/ml)
8 ml of ampicillin stock (100 mg/ml)
10 ml of L-SeMet stock (10 mg/ml)
10 ml of L-SeMet stock (10 mg/ml)
8 ml seed culture
8 ml seed culture


aliquote the 2 liter solution into 8X 250 ml  
aliquote the 2 liter solution into 8X 250 ml
grow at 37°C over night until OD600=0.400
grow at 37°C over night until OD600=0.400
induce protein expression  
 
induce protein expression
harvest cells 20 hours later
harvest cells 20 hours later


== M63 stock solution: ==
== M63 stock solution: ==
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15 g KH<sub>2</sub>PO<sub>4</sub>
15 g KH<sub>2</sub>PO<sub>4</sub>
45.6 g K<sub>2</sub>HPO<sub>4</sub>
45.6 g K<sub>2</sub>HPO<sub>4</sub>
10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
10 g (NH<sub>4</sub>)<sub>2</sub>SO<sub>4</sub>
2.5 ml FeSO<sub>4</sub> at 2.5 mg/ml
2.5 ml FeSO<sub>4</sub> at 2.5 mg/ml


== solution 1: ==
== solution 1: ==


for 50 ml (in ddH2O)
for 50 ml (in ddH<sub>2</sub>O)


add 200 mg of the following amino acids:
add 200 mg of the following amino acids:
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